PROF.

GUSTAVO DE-DEUS (Orcid ID : 0000-0001-7778-047X)

DR. FELIPE GONÇALVES BELLADONNA (Orcid ID : 0000-0001-9972-6861)
DR. ERICK MIRANDA SOUZA (Orcid ID : 0000-0003-2074-0834)
Accepted Article
DR. EMMANUEL JOÃO NOGUEIRA LEAL SILVA (Orcid ID : 0000-0002-6445-8243)

Article type : Original Scientific Article

Root dentinal microcracks: a post-extraction experimental phenomenon?

G. De-Deus1, D. M. Cavalcante1, F. G. Belladonna1, J. Carvalhal1, E. M. Souza2, R. T. Lopes3, M. A.

Versiani4, E. J. N. L. Silva5, P. M. H Dummer6

1
Department of Endodontics, Fluminense Federal University, Niterói, Rio de Janeiro, Brazil,
2
Department of Dentistry II, Federal University of Maranhão, São Luís, Maranhão, Brazil, 3Nuclear

Engineering Program, Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil,
4
Oral Health Center, Brazilian Military Police, , Minas Gerais, Brazil, 5Department of Endodontics,

Grande Rio University, Duque de Caxias, Rio de Janeiro, Brazil, 6School of Dentistry, College of

Biomedical and Life Sciences, Cardiff University, Cardiff, UK

Running title: Dentinal microcracks: an experimental phenomenon?

Keywords: cadaver model, dentinal defects, microcracks, micro-CT, , vertical root fracture.

Corresponding author:

Prof. Dr. Gustavo De-Deus

Av. Henrique Dodsworth 85 Apto 808 - Lagoa, Rio de Janeiro, RJ, Brazil, ZIP CODE: 22061-030

Phone: (55) 21 99700-8254

e-mail: endogus@gmail.com

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/iej.13058
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 Abstract

Aim To investigate the prevalence, location, and pattern of preexisting microcracks in non-
Accepted Article
endodontically treated teeth from fresh cadavers. Micro-computed tomography (micro-CT)

technology was used as the analytical tool enabling full screening of the root dentine with the teeth

retained in their original alveolar socket.

Methodology As a pilot study and to validate the present method, a series of 4 high-resolution scans

were performed on one bone-block specimen with teeth collected postmortem: (i) entire bone-block

including the teeth, (ii) second molar tooth extracted atraumatically from the bone-block (iii)

extracted tooth dehydrated to induce dentinal defects and (iv) entire bone-block following reinsertion

of the extracted tooth into its matching alveolar socket. In the main study, forty-two dentoalveolar

maxillary bone-blocks each containing 3-5 adjacent teeth (178 teeth in a total) were collected

postmortem and scanned in a micro-CT device. All cross-section images of the 178 teeth (n = 65,530)

were screened from the cementoenamel junction to the apex to identify the presence of dentinal

defects.

Results In the pilot study, the microcracks observable when the dehydrated tooth was outside the

bone-block remained detectable when the entire bone-block plus reinserted tooth was scanned. This

means that the screening process revealed the presence of the same microcracks in both experimental

situations (the tooth outside and inside the maxillary bone-block). From a total of 178 teeth in the

bone-blocks removed from cadavers, 65,530 cross-sectional images were analyzed and no dentinal

microcracks were detected.

Conclusions This in situ cadaveric model revealed the lack of preexisting dentinal microcracks in

non-endodontically treated teeth. Thus, the finding of dentinal microcracks observed in previous

cross-sectional images of stored extracted teeth is unsound and not valid. It should be assumed that

microcracks observed in stored extracted teeth subjected to root canal procedures are a result of the

extraction process and/or the post-extraction storage conditions. Therefore, As a consequence, the

presence of such dentinal microcracks in stored extracted teeth – observevable in cross-sectional

images of the roots - should be refered to as experimental dentinal microcracks.

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 Introduction

During the investigation of vertical root fractures (VRFs), the microstructural integrity of root
Accepted Article
dentine and cementum has been evaluated using destructive (tooth-sectioning) (Hin et al. 2013, Liu et

al. 2013, Arias et al. 2014, Ashwinkumar et al. 2014, Karataş et al. 2015, Saber & Schäfer 2016,

Bahrami et al. 2017, Kfir et al. 2017) and non-destructive (micro-computed tomography [micro-CT])

experimental models (De-Deus et al. 2014, 2015, 2016, 2017a, 2017b, Bayram et al. 2017,

PradeepKumar et al. 2017, Zuolo et al. 2017). Most of these studies used either teeth that had been

stored for varying periods of time (Hin et al. 2013, Liu et al. 2013, De-Deus et al. 2014, 2015, 2016,

2017a, Karataş et al. 2015, Bayram et al. 2017, Zuolo et al. 2017) or were freshly extracted

(Ashwinkumar et al. 2014, Saber & Schäfer 2016, Kfir et al. 2017, PradeepKumar et al. 2017) with

only a few studies being conducted using cadaveric models (Arias et al. 2014, Bahrami et al. 2017,

De-Deus et al. 2017b).

The use of a non-destructive high-resolution imaging technology, i.e. micro-CT, has made it

possible to gain a more reliable insight into the phenomenon of dentinal microcrack formation. Micro-

CT allows the internal structure of opaque objects (e.g. teeth) to be observed by screening hundreds of

slices per specimen, where the full extent of crack lines can be mapped (De-Deus et al. 2014, 2015,

2016, 2017a, 2017b, Bayram et al. 2017, PradeepKumar et al. 2017, Zuolo et al. 2017). The use of

micro-CT thus allows root dentine and cementum to be observed in their original state, i.e. after

extraction, and then examined again after root canal procedures. Based on this method, two main

conclusions have been drawn: (i) the lack of relationship between dentinal microcrack formation and

mechanical preparation of root canals with nickel-titanium (NiTi) instruments per se (De-Deus et al.

2014, 2015, 2016, 2017a, 2017b, Bayram et al. 2017, Zuolo et al. 2017) and (ii) the recognition of

preexisting microcracks as a phenomenon in untreated teeth (De-Deus et al. 2014, 2015, 2016, 2017a,

2017b, Bayram et al. 2017, PradeepKumar et al. 2017, Zuolo et al. 2017). Preexisting microcracks are

microstructural defects in roots of non-endodontically treated teeth with their aetiology being credited

to factors such as age, parafunctional stresses (Yang et al. 1995, Chan et al. 1998), or restorative

procedures (Kishen 2006, Shemesh et al. 2009).

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 Although rarely reported in destructive sectioning studies, preexisting microcracks have been

observed in non-endodontically treated specimens even in the initial studies that focused mostly on
Accepted Article
the relationship between the development of dentinal defects and root canal preparation techniques

(Arias et al. 2014, Karataş et al. 2015, Bahrami et al. 2017, Kfir et al. 2017). Interestingly, the use of

micro-CT technology in studies using stored teeth revealed a high incidence (ranging from 12.31% to

41.44%) of preexisting microcracks in the baseline images acquired from untreated teeth (De-Deus et

al. 2014, 2015, 2016, 2017a, Bayram et al. 2017, Zuolo et al. 2017). Actually, information provided

by untreated control groups have been significant and controversial. Most often, no microcracks can

be observed when stored sound teeth are sectioned horizontally (Shemesh et al. 2009, Karataş et al.

2015, Kfir et al. 2017), while in some studies using cadaveric models microcracks have been reported

in the untreated control groups (Arias et al. 2014, Bahrami et al. 2017). Conversely, a low prevalence

of preexisting microcracks was reported when evaluating freshly extracted teeth (7.1%)

(PradeepKumar et al. 2017) or in a cadaveric model (2.46%) (De-Deus et al. 2017b) when using

micro-CT technology. This means that the phenomenon of preexisting microcracks must be

reconsidered as a consequence of the new evidence provided by the micro-CT imaging method and

the use of either freshly-extracted teeth (De-Deus et al. 2017b, PradeepKumar et al. 2017) or teeth

within a cadaver model (De-Deus et al. 2017b).

In summary, the existence of preexisting microcracks has been controversial. The somewhat

puzzling occurrence of preexisting microcracks has created interest in potential aetiological factors as

well as in determining whether VRFs are preceded by such microstructural defects. Considering its

as-yet-unknown aetiology as well as the lack of knowledge on this phenomenon, the current study

aimed to investigate the prevalence, location, and pattern of preexisting microcracks in non-

endodontically treated teeth from fresh cadavers. Micro-CT technology was used as the analytical tool

enabling full screening of the root dentine with the teeth retained in their original alveolar socket. The

core hypothesis being tested was that preexisting microcracks occur at a high frequency in non-

endodontically treated teeth.

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 Materials and methods

Sample selection
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Forty-two dentoalveolar maxillary and mandibular bone-blocks, each containing 3-5 adjacent

teeth (a total of 178 teeth), were collected postmortem during the autopsy of several adult donors. The

family members gave their informed consent which was obtained under a research protocol approved

by the local Forensic Department and the National Committee on Health Research Ethics (protocol

no. 931.732). The age of the donors ranged from 19 to 44 years (mean age, 31 years). Inclusion

criteria were the presence of non-carious maxillary or mandibular first and second premolars and

molars surrounded by alveolar bone and periodontal ligament. Bone-blocks with teeth were stored at -

20 ºC and submitted to the experimental procedures within 40 days from their collection.

Before the scanning procedures, frozen bone-blocks were removed from the freezer and

placed into a refrigerator at a constant temperature of 8 ºC for a slow defrost. After 3-4 hours, each

bone-block was scanned in a micro-CT device (SkyScan 1173; Bruker-microCT, Kontich, Belgium)

using an isotropic resolution of 13.18 μm at 90 kV and 88 mA through 360º rotation around the

vertical axis, with a rotation step of 0.5º, camera exposure time of 1000 milliseconds, and frame

averaging of 5. The x-rays were filtered with a 1-mm-thick aluminum filter. The acquired images

were reconstructed into cross-sectional slices with NRecon v.1.6.10 software (Bruker-microCT) using

standardized parameters for beam hardening (15%), ring artifact correction (5), and contrast limits

(0.0095-0.03), resulting in the acquisition of 1300-1600 transverse cross-sections per bone-block.

Pilot study - validation method

Validation of the present method was based on 4 high-resolution micro-CT scans of a single

bone-block containing 3 teeth (one premolar, one first molar and one second molar) following the

same parameters previously described. The sequence of micro-CT scans was: (i) entire bone-block,

(ii) extracted tooth, (iii) dehydrated extracted tooth, and (iv) entire bone-block after reinsertion of the

extracted tooth into its alveolar socket (Fig. 1). The integrity of the dentine (presence of dentinal

microcracks) was evaluated by screening the cross-sectional images obtained in the reconstruction

step, from the cementoenamel junction to the root apex, by 3 blinded calibrated examiners. The

calibration process was based on viewing sessions using cross-sectioning images with previously

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 identified microcracks. The image analysis was repeated twice at 2-week intervals to validate the

process of microcracks identification.

Accepted Article
In the first scan, no microcracks were observed (Fig. 2 [a] and [b], and Fig. 3 [a] and [b]).

Then, the maxillary second molar tooth was atraumatically removed from the bone-block by avoiding

touching or damaging the surrounding tissues (Fig. 1 [c], [d] and [e]). This technique involved a

careful detachment of 2/3 of the roots with periotomes until luxation occurred and, to minimize

potential tooth damage, extraction forces were used only for tooth withdrawal, and not to loosen it.

The extracted molar was immediately scanned and the cross-sectional images were screened as

described above. No microcracks were observed in the second scan (Fig. 2 [c] and Fig. 3 [c]).

Aiming to induce the development of dentinal defects, the second molar tooth was submitted

to a dehydration process using a standard graded series of alcohols (50%, 60%, 70%, 80%, 90% and

100% ethanol). Then, the tooth was placed into an auto-desiccator cabinet (Bel-Art automatic

desiccator clear 2.0, Wayne, NJ, USA) and scanned on a weekly-basis to verify the presence of

microcracks. After a period of 3 months, the scan (third scan) clearly revealed the presence of dentinal

microcracks (Fig. 2 [d] and Fig. 3 [d]). Subsequently, the specimen was carefully reinserted in its

original alveolar socket and the entire bone-block was rescanned (fourth scan). The image analysis of

the cross-sections revealed that the microcracks observed when the tooth was outside the bone-block

remained detectable when the entire bone-block was scanned (Fig. 2 [e] and [f], and Fig. 3 [e] and

[f]).

Image analysis

Visualization and qualitative analysis of the reconstructed image stacks of the 42 bone-blocks

were assessed using CTVol v.2.3 software (Bruker-microCT). All cross-sectional images of the 178

teeth (n = 65,530) were screened from the cementoenamel junction to the apex to identify the

presence of dentinal defects. Three previously calibrated examiners, blinded to the experimental

design, screened all images at 2-week intervals. In case of divergence, images were evaluated together

until full agreement was reached (De-Deus et al. 2016).

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 Results

In the pilot study, microcracks observed when the tooth was outside the bone-block remained
Accepted Article
detectable when the entire maxillary segment was scanned, which validated the method of assessing

dentinal microcracks in a fresh cadaver model through micro-CT technology (Figs. 1 to 3).

From a total of 178 teeth in the bone-blocks removed from cadavers, 65,530 cross-section

images were analyzed and no dentinal microcracks were detected. Figs. 4 and 5 show representative

images from coronal, middle, and apical thirds of a selection of teeth evaluated in the study.

Discussion

In the current study, the incidence of dentinal microcracks in non-endodontically treated teeth

was assessed in situ through micro-CT images of 178 teeth in maxillary and mandibular bone-blocks

obtained from 42 fresh cadavers. No preexisting dentinal microcrack were observed, refuting the core

hypothesis. The absence of such dentinal microcracks in a methodology that is under close to in vivo

conditions – a human cadaver model - suggests that microcracks may occur due to post-extraction

manipulation or the storage conditions of the experimental teeth. This finding means that such

dentinal microcracks - observable in cross-sectional images of the roots - may not exist in the clinical

setting; in fact, thus far this type of dentinal defect has been observable only under post-extraction

experimental conditions (De-Deus et al. 2014, 2015, 2016, 2017a, Bayram et al. 2017, PradeepKumar

et al. 2017, Zuolo et al. 2017).

The present result contrasts with the accumulated knowledge regarding dentinal microcrack

formation that was been published since 2009 (De-Deus et al. 2014, 2015, 2016, 2017a, Bayram et al.

2017, PradeepKumar et al. 2017, Zuolo et al. 2017). Actually, the concept that dentinal microcracks

are a post-extraction experimental phenomenon are partially supported by recent insights on this

topic. Shemesh et al. (2018) reported the impact of environmental conditions on dentinal tissue and

demonstrated that loss of water produces stresses that are sufficient to induce spontaneous dentinal

defects, demonstrating experimentally that the biomechanical response of root dentine is highly

influenced by its degree of hydration. This is in accordance with previous findings that showed that

residual microstrain concentrations in hydrated roots was a controlled phenomenon and also that

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 dehydrated dentine had lower toughness (Jameson et al. 1993, Kahler et al. 2003. Kruzic et al. 2003)

and was more brittle (Huang et al. 1992). Thus, results reported in the study of Adorno et al. (2013)
Accepted Article
may be thought of as a consequence of tooth dehydration, since microcrack propagation continued in

root slices even after 1 month of storage, even though no further stress was applied to the dentine. In

this sense, the dehydration process that teeth endure outside of the oral environment may explain the

high prevalence (12.31% to 41.44%) of dentinal microcracks in the baseline images of non-

endodontically treated stored teeth evaluated through micro-CT technology (De-Deus et al. 2014,

2015, 2016, 2017a, Bayram et al. 2017, Zuolo et al. 2017), considering that the extracted teeth were

obtained from tooth banks that used a variety of storage conditions.

Another important study used micro-CT to evaluate the prevalence, location, and pattern of

preexisting dentinal microcracks in 633 freshly-extracted non-endodontically treated teeth and found

dentinal defects in 45 teeth (7.1% of the sample) (PradeepKumar et al. 2017). Similarly, De-Deus et

al. (2017b) using a cadaveric model also reported 2.46% of dentinal defects in the baseline images

from non-endodontically treated teeth. In accordance with the present results, these important

outcomes establish a more realistic prevalence of preexisting dentinal microcracks in extracted teeth,

in contrast with the substantial number of defects reported in previous micro-CT studies using stored

teeth (De-Deus et al. 2014, 2015, 2016, 2017a, Bayram et al. 2017, Zuolo et al. 2017). These findings

emphasise the low prevalence of preexisting microcracks and raise serious doubts about the validity

of most studies on dentinal cracks using extracted teeth, since cracks are likely to be a consequence of

the post-extraction experimental conditions. Based on this scientific evidence, it may be inferred that

dehydration of dentinal tissue is the main cause of microcracks in non-endodontically treated teeth

reported over the last decade. The prevalence of this phenomenon is thus a function of the interplay

between the origin of the specimen - storage versus freshly-extracted teeth/cadaveric model, and the

analytical method used - sectioning versus non-destructive micro-CT.

In a broader sense, the development of VRFs in endodontically-treated teeth is usually

attributed to factors such as age, the root and root canal anatomy, masticatory function and/or the

presence of excursive interferences or parafunctions that teeth could be subjected to during a patients’

life (Arias et al. 2014). However, it is possible that VRFs develop as a consequence of cracked or split

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 teeth initiated originally from the crown. Hence, it is important to consider the possible implications

of the conditions under which teeth are stored when analysing the results of laboratory studies since
Accepted Article
unintentional dehydration will introduce systematic experimental flaws, irrespective of care taken

during the rest of the experiment. This strongly suggests that in situ approaches such as models using

fresh cadavers should be considered as a gold standard to assess the behaviour of dentinal tissue in

terms of crack initiation and propagation.

The methodology used in the current study appears to be the close to an ideal experimental

model to study the phenomenon of microcracks and the overall status of the dentine. The use of an in

situ fresh cadaveric model, in which the bone and periodontal ligament remained preserved and also,

the viscoelastic properties of the attachment apparatus, together with a highly accurate and non-

destructive imaging method (micro-CT) for the assessment of the integrity of dentinal tissue has clear

advantages over other methodologies used previously in the study of dentinal defects, that is,

sectioning and micro-CT analysis of stored teeth. Furthermore, the cadaveric model avoids the impact

of tooth extractions and thus the use of periotomes, luxators or forceps, which usually are suggested

as generators of dentinal defects. However, it is necessary to emphasise that the sampling used in the

current study has one limitation, the age range of the cadavers which was between 19 and 44 years

old. Thefore, future work should focus on assessing the presence of dentinal defects in older cadavers.

As stated in the first study on dentinal microcracks using a cadaveric model and micro-CT

(De-Deus et al. 2017b), there is no international agreement, general regulations, or standards of tissue

banking on a specific storage temperature for teeth inside bone-blocks. A statement from the

American Association of Tissue Banks (2008) recommended a storage temperature of -20 °C for up to

6 months and -40 °C for longer periods of deep frozen preservation. However, the influence of storage

time and freezing temperatures on the biomechanical properties of teeth are not entirely understood

and are still to be determined. In the present study, the storage temperature of the cadaveric bone-

blocks followed that used by De-Deus et al. (2017b) and did not affect the structure of the bone or

teeth, which was -20 ºC, as recommended, with a period of slow defrost before scanning and further

experimental procedures.

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 Concerns regarding whether the scanning resolution of micro-CT images may or may not be

sufficient to detect smaller microcracks have also been raised (Pop et al. 205, De-Deus et al. 2016,
Accepted Article
PradeepKumar et al. 2017). Nevertheless, the validation of the micro-CT imaging method for the

observation of the dentinal defects in extracted teeth has already been reported (De-Deus et al. 2016);

it was demonstrated that defects visualized by direct observation of dentine with reflected light

microscopy (using the sectioning method) are also visualized in the reconstructed cross-sectional

images obtained by high-resolution micro-CT scans. However, the same is not necessarily valid when

the teeth are scanned within cadaveric bone-blocks. Therefore, due to the innovative character of the

observation of the dentinal defects in the images scanned from cadaveric bone-blocks, the validation

of the method was necessary to eliminate any possibility of false-negative results. The results revealed

that the screening process was able to demonstrate the presence of the same microcracks in both

experimental setups (tooth outside and inside the maxillary bone-block), validating the method to

assess dentinal microcracks in a fresh cadaver model through micro-CT technology.

The result of this study suggests that future works should focus on the existence of dentinal

root dentinal microcracks in non-endodontically treated teeth. In the meantime, until proven

otherwise, it shoud be assumed that dentinal microcracks observed in stored extracted teeth subjected

to root canal procedures are in fact a result of the extraction process and/or the post-extraction storage

conditions. As a consequence, the presence of such dentinal microcracks in stored extracted teeth -

observevable in cross-sectional images of the roots under experimental conditions - should be refered

to as experimental dentinal microcracks.

Conclusions

This in situ cadaveric model revealed the absence of preexisting dentinal microcracks in non-

endodontically treated teeth. This means that the prevalence of dentinal microcracks observed in

previous cross-sectional images of stored extracted teeth is flawed. It also questions whether

microcracks - observable in cross-sectional images of roots in extracted teeth really occur in non-

endodontically treated teeth in the clinical setting.

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 Conflict of Interest statement

The authors have stated explicitly that there are no conflicts of interest in connection with this article.
Accepted Article
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 Figure legends
Accepted Article
Figure 1 Sequence illustrating the atraumaticaly extraction of a molar tooth used in the validation

process. (a, b) 3D model and image of a maxillary bone-block of a fresh cadaver. (c, d) The

atraumatic technique involved the careful detachment of 2/3 of the roots with periotomes until

luxation occurred. (e) extracted second molar.

Figure 2 (a) Typical cross-sectional image of a maxillary bone-block of a fresh cadaver. The molar

tooth is visualized inside the alveolar bone socket. No microcracks are observed. (b) Detail of the

distal-buccal (DB) root of the second molar underlining the integrity of the root dentine, where no

microcracks are observed. (c) Cross-sectional image of the second molar tooth immediately after

tooth removal. No microcracks are observed. (d) Cross-sectional image of the extracted second molar

tooth scanned 3 months after the controlled dehydration process - a microcrack was induced and

clearly observable in the DB root of the second molar tooth (arrow). (e) Cross-sectional image of the

second molar tooth after reinsertion in its original place into the alveolar bone socket. The induced

microcrack is clearly observable in the DB root of the second molar tooth (arrow). (f) Detail of the

DB root of second molar underlining the presence of the microcrack (arrow) that was not present in

images (a) and (b).

Figure 3 Typical cross-sectional image of a maxillary bone-block of a fresh cadaver. The molar tooth

is visualized inside the alveolar bone socket. No microcracks are observed. (b) Detail of the distal-

buccal (DB) root of the second molar underlining the integrity of the root dentine, where no

microcracks are observed. (c) Cross-sectional image of the second molar tooth immediately after

tooth removal. No microcracks are observed. (d) Cross-sectional image of the extracted second molar

tooth scanned 3 months after the controlled dehydration process - a microcrack was induced and

clearly observable in the DB root of the second molar tooth (arrow). (e) Cross-sectional image of the

second molar tooth after reinsertion in its original place into the alveolar bone socket. The induced

microcrack is clearly observable in the DB root of the second molar tooth (arrow). (f) Detail of the

This article is protected by copyright. All rights reserved.

 DB root of second molar underlining the presence of the microcrack (arrow) that was not present in

images (a) and (b).

Accepted Article
Figure 4 Cross-sectional images of roots from a maxillary bone-block containing pre-molars and

molars teeth where it is possible to observe the status of the dentinal tissue without any dentinal

defect.

Figure 5 Cross-sectional images of roots from a maxillary bone-block containing pre-molars and

molars teeth where it is possible to observe the status of the dentinal tissue without any dentinal

defect.

This article is protected by copyright. All rights reserved.

Accepted Article

This article is protected by copyright. All rights reserved.

Accepted Article

This article is protected by copyright. All rights reserved.

Accepted Article

This article is protected by copyright. All rights reserved.